Optimization of Solid-State Fermentation with Lactobacillus brevis and Aspergillus oryzae for Trypsin Inhibitor Degradation in Soybean Meal

The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.

Spore production in the solid-state fermentation of stevia residue by Trichoderma guizhouense and its effects on corn growth

Trichoderma is an important and widely used plant growth-promoting fungus(PGPF).In this study,stevia residue amended with amino acids hydrolyzed from animal carcasses was used for the production of Trichoderma guizhouense NJAU 4742 by solid-state fermentation,and then its potential to promote corn plant growth was evaluated in combination with chemical fertilizer(CF)or organic fertilizer(OF).The highest spore number of 7×10^(9) CFU g^(–1) fresh weight was obtained under the following optimal parameters:material ratio of 50%(stevia residue:rice bran=1:1),pH value of 3.0(amended with 6.67%amino acids),initial moisture content of 60%,inoculum size of 10%,material thickness of 3 cm and an incubation time of 4 days.The aboveground corn plant biomass obtained with T.guizhouense applied alone and with CF treatments were slightly higher than those of no fertilizer control and CF treatments,respectively.However,T.guizhouense applied with OF significantly(P<0.05)increased aboveground biomass compared to OF and yielded the highest aboveground biomass among all the treatments.Moreover,T.guizhouense applications primarily influenced the fungal bulk soil community composition,among which three OTUs(OTU_(2) and OTU_(9) classified as Chaetomium,and OTU_(4)classified as Trichoderma)were stimulated in both bulk and rhizosphere soil.Notably,a specific OTU_(3)(Phymatotrichopsis)was only stimulated by T.guizhouense applied with OF,possibly leading to high soil productivity.These results show that it is feasible to employ stevia residue in the eco-friendly fermentation of T.guizhouense,which is strongly suggested for enhancing OF applications.

The variation of two extracellular enzymes and soybean meal bitterness during solid-state fermentation of Bacillus subtilis

The debittering effect of extracellular enzymes from Bacillus subtilis ACCC 01746 was studied using soybean meal as a substrate for solid-state fermentation(SSF).Results showed that B.subtilis produces proteases and carboxypeptidase in the early stage of SSF(0–8 h).Proteases are dominant and can hydrolyze the soybean protein into long-chain peptides with mild bitterness.Carboxypeptidase production is dominant at 8–16 h SSF,at which point soybean protein is further hydrolyzed and bitterness is enhanced.The strain then produces additional carboxypeptidase after 16 h,and bitterness is reduced.We compared the amino acid composition of the hydrolysates from soybean protein isolates to that of the fermented liquid of SSF.In the hydrolysates from soybean protein isolates that exhibit strong bitterness,62.81%of amino acids are hydrophobic and occur in the form of peptides.In the fermented liquid from soybean meal,16.22%of amino acids are hydrophobic and are mainly present in the form of free amino acids.The bitterness of fermented soybean hydrolysate is reduced from 5 to 0 when fermented for 24 h,suggesting that B.subtilis can effectively reduce bitterness,possibly due to the carboxypeptidase.Enzyme analysis shows that B.subtilis excretes carboxypeptidase during growth.The amino acids phenylalanine,alanine,tyrosine,and leucine at the C-terminal of the soybean bitter peptides in hydrolysates are cleaved in the presence of carboxypeptidase,resulting in complete debitterness.

Novel Protease from Aspergillus tamarii URM4634: Production and Characterization Using Inexpensive Agroindustrial Substrates by Solid-State Fermentation

This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.

Direct Solid-State Fermentation of Soybean Processing Residues for the Production of Fungal Chitosan by Mucor rouxii

The feasibility of utilizing soybean-processing residues such as soybean meal and hulls as substrates for chitosan production by the fungus Mucor rouxii ATCC 24905 via solid-state fermentation (SSF) was investigated. The effects of the type of soybean-based substrate, length of cultivation period, substrate moisture content, substrate pH, incubation temperature and extraction conditions on chitosan yield were determined. The results showed that a maximum fungal chitosan yield of up to 3.44% by dry substrate weight (34.4 g/kg) could be achieved using a pure soybean meal substrate with an initial moisture content of 50% (w/w) and pH of 5 - 6 incubated for six days at 25&degC. A more severe heat treatment (autoclaving vs. refluxing) resulted in higher chitosan extraction yields regardless of the strength of extraction reagents. Fourier transform infrared (FTIR) analysis of the fungal chitosan revealed its degree of deacetylation (DDA) to be between 55% and 60%.

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from <i>Bacillus thuringiensis</i>subsp. <i>kurstaki</i>

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt);and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h;while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores;malachite green-saffranin stained both δ-crystals and endospores;and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

Study on Temperature Gradients and Protein Enrichment by <i>Aspergillus oryzae</i>in Solid-State Fermentation on Packed Bed Bioreactor Using Jowar (Sorghum) Straw as Substrate

The packed bed solid state bioreactor designated as PBSSB is constructed in the present study. The experiments are carried out in packed bed bioreactor with jowar straw and inoculated with Aspergillus oryzae. Temperature gradient has been measured at different axial positions. It is found that the organisms grew rapidly during the period from 20 to 30 h during which heat generation is more. These results are in agreement with other researchers. The fermented jowar straw shows threefold increase in protein content. This can be utilized as high value nutritional feed to animals.

Protein Enrichment of Potato Peels Using Saccharomyces cerevisiae via Solid-State Fermentation Process

In order to add value to potato peels and also curb their environmental pollution problems, this study investigated the protein enrichment of potato peels with Saccharomyces cerevisiae via Solid-State Fermentation (SSF). SSF is a fermentation process which involves solid matrix and is carried out in absence or near absence of free water. SSF of potato peel mashed was carried out with S. cerevisiae at 30°C, pH of 5.5, moisture adjustment between 40 and 90%, addition of ammonium sulphate and urea salts as nitrogen supplements for the microorganisms for 3 days. The results showed that the percentage crude protein content of all the fermented samples increased significantly when compared with the unfermented sample. 40% moisture content adjustment and ammonium sulphate as nitrogen source gave the best result. The crude protein increased from 12.5% to 21.86%, which is 74.88% increment for ammonium sulphate supplementation, and 12.5% to 18.42%, which is 47% increment for urea supplementation. Therefore, the fermented peels could serve as good source of cheap protein enriched feed for livestock.

Optimization of the Chitinase Production by Different Metarhizium anisopliae Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control various insect pests. These fungi attack the integument of the host using an enzymatic complex. Among the enzymes found in this complex, chitinase is an important component. However, the relation between the chitinase production and the virulence from different M. anisopliae strains has not been analyzed. In this manuscript it is presented the chitinase production by four M. anisopliae strains with different potential of virulence in Solid-State Fermentation using silkworm chrysalis as substrate. The higher chitinase level was obtained with the strain IBCB 360 (7.14 U/g of substrate) with potential virulence of 68% on Diatrea saccharalis. The enzyme production was optimized for all strains using a factorial planning (CCRD) considering the cultivation time and medium humidity as independent variables. The maximal production of chitinase was obtained at a range from 8 to 12-days old cultures and from 45% to 62% of moisture according to the surface response plot, with high R2 value. The enzyme production by the strain IBCB 167 was increased two-folds under optimized conditions, while for the strains IBCB 360 and 425 the chitinase production was increased four-folds and nine-folds for the strain IBCB 384.

Optimization of Parameters for the Production of Lipase from Pseudomonas sp. BUP6 by Solid State Fermentation

Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strain BUP6 on agro-industrial residues. Pseudomonas sp. strain BUP6 showed higher lipase production when grown in Basal salt medium (BSM) supplemented with oil cakes. Initially, five different oil cakes (obtained after extracting oil from coconut, groundnut, cotton seed, gingelly or soybean) were screened to find out the most suitable substrate-cum-inducer for the production of lipase. Among them, groundnut cake supported the maximum production of lipase (107.44 U/gds). Box-Behnken Design (BBD), followed by response surface methodology (RSM) was employed to optimize the culture parameters for maximizing the production of lipase. Using the software Minitab 14, four different parameters like temperature, pH, moisture content and incubation time were selected for the statistical optimization, which resulted in 0.7 fold increase (i.e., 180.75 U/gds) in production of lipase under the optimum culture conditions (temperature 28&#176C, pH 5.9, moisture 33% and incubation 2 d). Thus, this study signifies the importance of SSF for the production of industrially-significant lipase using agro-industrial residues as solid support.

Physicochemical Properties and Bioactivities of Rice Beans Fermented by Bacillus amyloliquefaciens

The purpose of this study was to investigate the physicochemical properties and bioactivities of rice beans(Vigna umbellata)fermented by Bacillus amyloliquefaciens.The fermentation conditions were optimized on the basis of the fibrinolytic activity.Under the optimal fermentation conditions,the fibrinolytic activity reached a maximum of 78.0 FU·g^(-1)(4890 IU·g^(-1),fibrin plate method,FU:fibrin degradation unit).The contents of peptides(which increased from 2.1 to 10.9 g per 100 g),total phenolics(from 116.7 to 388.5 mg gallic acid per 100 g),total flavonoids(from 235.5 to 354.3 mg rutin per 100 g),and anthocyanin(from 20.1 to 47.1 mg per 100 g),as well as the superoxide dismutase activity(from 55.3 to 263.6 U·g^(-1))in rice beans were significantly increased after fermentation.The 2,2-diphenyl-1-picrylhydrazyl(DPPH)and 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt(ABTS)free radical scavenging activities and ferric reducing antioxidant power(FRAP)of fermented rice beans were 1.9–4.8 times higher than those of unfermented rice beans.Moreover,fermentation induced an increase in the dipeptidyl peptidase IV(DPP-IV)inhibition,a-glucosidase inhibition,and anticoagulant activities of rice beans.Rice beans fermented by Bacillus amyloliquefaciens may serve as a functional food with potential benefits for the prevention of thrombotic diseases.

Application and optimization of solid-state fermentation process for enhancing polygalacturonase production by Penicillium expansum

Penicillium expansum 3.5425 was applied in solid-state fermentation(SSF)of agricultural wastes for polygalacturonase biosynthesis.Among various carbon additives,apple pomace was most suitable for the biosynthesis of polygalacturonase(1440.57 U/g).Optimization of medium parameters using rotational orthogonal design(ROD)experiment combined with optimal fermentation conditions resulted in a 2.72-fold increase in the polygalacturonase production.By using ammonium sulphate precipitation,ion-exchange and gel-permeation chromatography,the polygalacturonase produced by P.expansum 3.5425 was finally purified which had specific activity of 19269 U/mg and molecular weight of 30 kDa.The enzyme was remarkably active in the pH range of 3-5 and at 50℃,which makes it more acceptable in the industrial application.Besides,partially purified polygalacturonase(875.15 U/mL)was used for apple juice clarification and the clarity at 0.4 mL/kg was maximum,which reveals a great potential of polygalacturonase in food industry.

Improvement of quality of Ginkgo biloba seeds powder by solid-state fermentation with Eurotium cristatum for developing high-value ginkgo seeds products

In this study,we investigated the feasibility of Ginkgo biloba seeds powder by solid-state fermen-tation with Eurotium cristatum for developing high-value ginkgo seeds products.The optimum fermentation medium was consisted of 10 g of 40-mesh ginkgo seeds powder loaded in 100 mL Erlenmeyer flask with 50%(w/w)of water content,4%(w/w)of MgSO_(4)and 5%(w/w)of KH_(2)PO_(4)addition.The optimum fermentation conditions were pH 5.0,2×10^(8)CFU/g of inoculum size,3 mL of sterilized water supplemented every two days during the four days of fermentation.Through fermentation,the spore number of E.cristatum was improved by about 36 times with the produc-tion of lovastatin reaching(54.10±0.16)μg/g.The antioxidant activity of fermented ginkgo seeds powder also got obvious enhancement,which could help eliminate excess free radicals produced by normal metabolism.The content of free amino acids increased by 82.32%.Except that the sugar was consumed in some degree,the other nutritional and functional components were well preserved while the content of detrimental ginkgolic acids was reduced by 44.97%.In addition,fermented ginkgo seeds powder possessed better digestibility and showed pleasant orange-like smelling.In conclusion,the quality of ginkgo seeds powder was remarkably improved through solid-state fermentation by using E.cristatum,which could be a promising way for functional applications of ginkgo seeds.

Solid-state fermentation technology and innovation for the production of agricultural and animal feed bioproducts

It has now passed more than forty years since solid-state fermentation(SSF)research developments have gained importance for the scientific community.After so many years,numerous processes and equipment for SSF were studied and designed focusing on the production of different commercially relevant bioproducts such as enzymes,fermented food,such as Chinese daqu and koji,organic acids,pigments,phenolic compounds,aromas,biosorbents and so many others.However,no review paper has been focused yet specifically on agricultural and animal feed bioproducts obtained through SSF techniques.This review comprises the description of agricultural sub-products that have been employed in most important developed pro-cesses concerning the production of animal feed products and agricultural products such as spores,probiotics,biofungicides,bioinsecticides and other biopesticides,biofertilizers and plant growth hormones.Major designed SSF bioreactors are also described and the most important related cases of successful employment of the technique are reported.Finally,a summary of patents and innovations regarding SSF products and processes in this area is presented,showing that the main involved countries are China,South Korea,India and the USA.It is clear that the interest in this theme is increasing and that scientific and technological developments are still needed.

Bacteriostatic effect of lipopeptides from Bacillus subtilis N-2 on Pseudomonas putida using soybean meal by solid-state fermentation

Bacillus subtilis N-2 which was isolated from natto,produced lipopeptides using soybean meal as a substrate.This work aimed to purify,identify,and determine the antibacterial mechanism of lipopeptides produced by B.subtilis N-2.The fermented product obtained by solid-state fermentation was subjected to water extraction,acid precipitation,and methanol extraction.Fractions were separated and collected using a two-step ultrafiltration method and then identified by LC-MS/MS.Mass spectrometry characterization revealed the presence of four variants of iturin A that differed according to the p-amino fatty acid chain from C14 to C17 as well as the amino acid positions.A new lipopeptide(m/z 1070.3)was identified and its structure was different from the previously reported lipopeptides.The lipopeptides were shown to inhibit the growth of an isolate of Pseudomonas putida,a common pathogen in decaying fish,by changing membrane permeability.These results suggest that the lipopeptides from B.subtilis N-2 could be used as a biocontrol agent in aquaculture.

Highly effective extraction of oil from soybean by pretreatment of solid-state fermentation with fungi

The solid-state prefermentation by Aspergillus niger(CICC 2377)and Aspergillus flavus(CICC 40536)was employed to increase the oil extraction yield from soybean.The influence of incubation time on oil yield was investigated.The maximum oil yield extracted from the substrate prefermented by Aspergillus niger for 96 h was 23%,which increased by 47.4%compared with control(15.6%).In the same fermentation conditions,the maximum oil yield extracted from substrate prefermented by Aspergillus flavus was 21.6%,which increased by 38.5%compared with control(15.6%).The quality of soybean oil was not changed obviously by the pretreatment of fermentation with fungi.

Study on microbial protein and the mechanism of solid-state fermentation with periodical dynamic changes of air

The effect of different extraction and purification conditions on the microbial protein obtained from solid-state fermentation(SSF)and the effect of periodical dynamic changes of air on protein have been studied.The mechanism of solid-state fermentation with periodical dynamic changes of air is also discussed.Compared with static solid-state fermentation,periodical dynamic changes of air afford a higher protein mass;from 1 g of the fermentation microbe,5.3 mg of the intracellular protein(an increase of 34.63%)and 9.09 mg of the extracellular protein(an increase of 17.8%)were obtained on the sixth day of fermentation.The filter paper activity and carboxymethyl cellulose activity(FPA and CMCase)of the extracellular protein are 1.739μmol/s and 109.592μmol/s,respectively,which represent increases of 60.1%and 21.2%over the corresponding values for static solid-state fermentation.The FPA and CMCase of the intra-cellular protein are 0.245μmol/s and 6.392μmol/s,respec-tively,which represent decreases of 22.2%and 38.7%over the corresponding values for static solid-state fermentation.The enzyme activity of the microbial extracellular protein in solid-state fermentation with periodical dynamic changes of air on the fifth day is nearly equal to that on the sixth day without periodical dynamic changes of air,so the period of fermentation can be shortened.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)experiments suggest that pulsating air pressure stimulation leads to a decrease in the amount of protein component with molecule mass of about 80400Da,and an increase in the amount of protein component with molecule mass of about 28520Da.

Efficient production of poly-γ-glutamic acid by Bacillus velezensis via solid-state fermentation and its application

A novel glutamate-dependent poly-γ-glutamic acid(γ-PGA)-producing strain Bacillus velezensis CAU263 isolated from Chinese traditional Douchi was evaluated.An efficient method ofγ-PGA production was performed by this strain via solid-state fermentation(SSF)using guar meal.The maximal yield ofγ-PGA reached 158.5 g/kg dry weight(DW)in 250 mL flasks and 155.1 g/kg DW in shallow tray,respectively.The molecular weight ofγ-PGA was 3.8×106 Da,the ratio of L/D-glutamic acid was 26.1%and 73.9%.Additionally,γ-PGA significantly improved the operating characteristics of wheat dough,the specific volume of bread was increased by 15.8%and the bread hardness was reduced by 44.2%owing to the addition of 0.3 g/kgγ-PGA.Thus,γ-PGA could be produced by Bacillus velezensis CAU263 at high level using guar meal and might have potential application prospect in food industry.

Solid-state fermented plant protein sources in the diets of broiler chickens:A review

Protein sources are the second most important component in poultry diets.Due to the fluctuation in price of soybean meal(SBM) and persistent increase in feed prices,nutritionists have been exploring alternative protein sources.Replacement of SBM with alternative protein sources in poultry diets could reduce human-live stock competition for soybean and support the production of more animal protein.However,the use of alternative protein sources is limited to low inclusion due to the presence of antinutritional factors(ANF) such as glucosinolates(rapeseed meal),gossypol(cottonseed meal),nonstarch polysaccharides(NSP) in lupin flour,high fibre(palm kernel cake),total phenolic contents and phytic acid(canola meal) known to impair animal performance,nutrient digestibility and feed utilization.As a processing technique,solid-state fermentation(SSF) has been researched for a long time in the food industry.An important objective of SSF is the production of enzymes,organic acids and other metabolites of economic importance.In recent times,SSF has been employed to enhance nutrient bioavailability,inhibit gut pathogenic bacteria and reduce ANF in plant protein sources resulting in improved nutrient digestibility,thereby improving performance and gut health of broiler chickens.Unlike pigs,there is still a dearth of information on feeding solid-state fermented feed ingredients to broiler chickens.This review aims to describe the nutritional value of the solid-state fermented products of rapeseed meal,canola meal,cottonseed meal,palm kernel cake and lupin flour on performance and intestinal health of broiler chickens.

Advances in research on solid-state fermented feed and its utilization:The pioneer of private customization for intestinal microorganisms

With sustainable development of biotechnology,increasing attention has been placed on utilization of solid-state fermented feed(SFF).Solid-state fermented feed has been a candidate strategy to alleviate the contradiction between supply and demand of feed resources,ensure food hygiene safety,promoting energy conservation,and emission reduction.In production of SFF,a variety of organic acids,enzymes,vitamins,peptides,and other unknown growth factors are produced,which could affect performance of animals.Solid-state fermented feed produced by different fermentation techniques has great instability on different physiological stages of different animals,which hinders the application and standardized production of SFF.Herein,we summarize the current advances in the role of the characteristics of SFF prepared by different manufacturing technique and its research progress in animal experiments on growth performance,gastrointestinal ecology,and immune system,so as to provide references for further acquiring a relatively perfect set of SFF production and evaluation systems.